Dentinogenesis

Question 1

General concepts

Answer 1

1. Preodontoblasts differentiate from cells of dental papilla and form a layer of polarized cells along the inner enamel epithelium
   
   1. Its ability to repair or remodel dentin is fairly limited but some is certainly possible.
2. Odontoblasts start to deposit dentin (appositional growth) first and then enamel secretion starts (which is also when basal lamina starts to disappear)

Question 2

Odontoblast

Answer 2

1. Odontoblast is a factory depositing organic matrix and mineral
2. The ectomesenchymal cells adjoining th acellular zone rapidly enlarge and elongate to become preodontoblasts first and then odontoblasts as their cytoplasm increases in volume to contain increasing amounts of protein-synthesizing organelles.
3. The acellular zone between the dental papilla and the inner enamel epithelium gradually
   is eliminated as the odontoblasts differentiate and
   increase in size and occupy this zone. These newly differentiated cells are characterized by being highly polarized, with their nuclei positioned away from the inner enamel epithelium.
4. The matrix first accumulates as an unmineralized layer, predentin (PD), which gradually mineralizes to form mantle dentin
5. Odontoblasts are connected via tight and gap junctions, making the odontoblast layer semipermeable.
6. Odontoblasts are connected via intercellular junctions i.e. Junctional Complex separating the environment in which matrix deposition and mineralization take place
   
   1. This separation is selective, some molecules can pass through while others do not
   2. These junctions allow for concentration gradient of different materials and minerals away from other parts of the body; its leaky though
7. Gap junctions are involved in the intercellular signaling
8. Odontoblasts contain large secretory apparatus including well developed golgi apparatus and rough ER.
9. Secondary tubule connect between primary tubules

Question 3

Odontoblast Processes

Answer 3

1. Odontoblast processes transport proteins and ions from odontoblast body to the mineralization front and mineralized dentin
2. The processes contain secretory granules transporting materials from the cell body deep into dentin and also endocytose materials from dentin
3. Secretory vesicles contain proteins and other materials, migrate from the cell body via dentinotubules and arrive at other locations
   
   1. When dentin is dead, they change physical properties and is common due to complications with endodontic treatment

Question 4

Odontoblast life cycle stages

Answer 4

1. Preodontoblast
   
   1. Contain centrioles that are responsible for the growth of the cell
   2. Multiple processes are seen but only one of them become the actual odontoblast process
2. Secretory
3. Transitional
4. Aged

Question 5

Dentin: structure and composition

Answer 5

1. Dentin is the major tissue of a tooth
2. Mineralized collagenous tissue similar to bone
3. Mineral content is higher than in bone: ~70% by weight vs. 60-65% by weight in bones

Question 6

Mantle Dentin

Answer 6

1. Crown mantle dentin does NOT exist in the root area, only in the crown area instead directly adjacent to enamel
2. Crown circumpalpal dentin is the bulk dentin directly below the relatively ‘thin’ layer of mantle dentin
3. Mantle dentin is a thin layer of dentin (~20 um) at the interface with enamel near DEJ.
4. It is the first dentin layer to form
5. These fibers originate deep among the odontoblasts,
   extend toward the inner enamel epithelium, and fan
   out in the structureless ground substance immediately below the epithelium
6. Contains bundles of thick collagen fibrils running normal (perp.) to the dentin-enamel interface, so called von Korff fibrils, embedded in the matrix of thinner randomly oriented collagen fibrils
   
   1. von Korff (collagen) fibrils penetrate into the enamels and are responsible for robust interaction between dentin and enamel
7. Mantle dentin is morphologically distinct outer layer of dentin adjacent to enamel
   
   1. The first mineral phase in mantle dentin, amorphous calcium phosphate is localized in the matrix vesicles
   2. There are very few dentinal tubules in mantle dentin
   3. Instead, matrix vesicles are budding from the cell membrane of odontoblasts (which is moving toward pulp as it secretes new predentin) and help to mineralize the matrix
   4. The mineral phase first appears within the
      matrix vesicles as single crystals believed to be seeded by phospholipids present in the vesicle membrane. These crystals grow rapidly and rupture from the confines of the vesicle to spread as a cluster of crystallites that fuse with adjacent clusters to form a continuous layer of mineralized matrix.

Question 7

Dentinal tubules in mantle dentin

Answer 7

1. Dentinal tubules in mantle dentin branch & penetrate into enamel
2. These tubules transport materials to nurture and provide nutrient for maintaining the interface between enamel and dentin
3. Below DEJ, fibrils are randomly oriented; tubules in mantle dentin do NOT contain peritubular dentin

Question 8

Dentinal Tubules

Answer 8

1. Dentinal tubules run through dentin from the DEJ to the pulp cavity
2. Tubules branch close to the DEJ
3. Tubules contain odontoblast processes and nerve endings
4. No synaptic connections have ever been observed between these cells (odontoblast process & nerve)

Question 9

Sclerosis of dentin

Answer 9

1. When the tubule becomes filled with mineral it is called sclerotic
2. Sclerosis may be hastened by pulpal irritation
   
   1. Due to aging or injury; defense mechanisms to slow down caries so as to slow down propagation of further decay

Question 10

Circumpalpal Dentin - main dentin layers

Answer 10

1. Circumpalpal dentin comprises the bulk of dentin
2. Intertubular dentin comprised of mineralized collagen fibrils oriented in planes parallel to DEJ and normal to tubules
3. Peritubular dentin is highly mineralized but with NO collagen
4. Intertubular dentin
   
   1. Intertubular dentin is a bone-like tissue comprised of mineralized collagen fibrils in which crystals are organized into parallel arrays
5. Peritubular dentin
   
   1. A thin dense layer of dentin surrounding the tubules
   2. Lacks collagen
   3. Higher mineral content (40% more mineral than in intertubular dentin, very stiff)
   4. Despite lack of collagen mineral, crystallites in dentin organize into parallel arrays, similar to mineralized collagen fibrils

Question 11

Globular & Interglobular dentin

Answer 11

1. Globular dentin is formed due to uneven mineralization pattern at the mineralization front thus the interface between the predentin and dentin is NOT linear.
2. The pattern of mineralization persists in mineralized dentin
3. Compared to globular denin, interglobular dentin is undermineralized
4. Dentinal tubules can be seen running through both types
5. Interglobular dentin does NOT have peritubular dentin
6. Globular dentin is best seen in the mantle dentin region, where matrix vesicles give rise to mineralization foci that grow and coalesce.
7. In circumpulpal dentin the mineralization front can progress in a globular or linear pattern.
8. The size of the globules seems to depend on the rate of dentin deposition, with the largest globules occurring where dentin deposition is fastest. When the rate of formation progresses slowly, the mineralization front appears more uniform and the process is said to be linear.

Question 12

Primary curvatures of the tubules

Answer 12

1. Tubules in many locations of dentin may not run straight; they change directions, forming an S-like pattern consisting of 2 bends
2. The primary curvatures are mechanically important features that can withstand pressure and give additional elasticity to the tissue; like springs or shock absorbers

Question 13

Secondary curvatures in dentin

Answer 13

1. Secondary curvatures are small undulations of the dentinal tubules formed by periodic changes in the direction of the odontoblast process as the dentin matrix mineralized

Question 14

Incremental Lines of von Ebner

Answer 14

1. Lines of von Ebner occur at frequent intervals and represent small increments of dentin deposition, possibly reflecting a diurnal (daily) cycle of matrix deposition

Question 15

Contour lines of Owen

Answer 15

1. Contour lines of Owen are incremental lines in dentin representing longer increments of dentin deposition at sites of more pronounced secondary curvature
2. Caused by serious systemic physiological changes; i.e. diseases, changes in diet, seasonal changes, etc.
3. The most accentuated contour line is the neonatal line

Question 16

Primary and Secondary dentin

Answer 16

1. Primary dentin comprises the bulk of dentin, deposited during the appositional stage. However, once the roots are formed, the dentin deposition is not completely ceased.
2. Instead, slow and not uniform dentin deposition continues, which is now called secondary dentin. It is less organized and the directions of the tubules in it are different from primary dentin; mineralization after the completion of the roots
3. Secondary dentin is deposited after root formation is completed, is formed by the same odontoblasts that formed primary dentin, and is laid down as a continuation of the primary dentin. Secondary dentin formation is achieved in essentially the same way as primary dentin formation, although at a much slower pace.

Question 17

Root dentin

Answer 17

1. Root dentin forms similarly to coronal dentin, but some differences have been reported. The outermost layer of root dentin, the equivalent of mantle dentin in the crown, shows differences in collagen fiber orientation and organization, in part because the collagen fibers from cementum blend with those of dentin
2. Does NOT contain any peritubular dentin

Question 18

Granular Layer of Tomes

Answer 18

1. Forms at the interface between dentin and cementum
2. can be seen just below the surface of the dentin where the root is covered by cementum
3. The origin is not clear but might be involved in the formation of interface between dentin and cementum

Question 19

Transparent dentin

Answer 19

1. Transparent dentin is most frequently observed in the root
2. The tubules of transparent dentin are sclerotic
3. Dentin appears transparent when cleared with a solvent, which obviously means its dead
4. When dry, transparent dentin is opaque.

Question 20

Pathological dentin types

Answer 20

1. Tertiary dentin forms in response to severe damage of cells by trauma or infection. The types depend on the strength of the stimulus and the source of cells forming the dentin
   
   1. Reactionary dentin forms by injured odontoblasts as a reaction to injury
   2. Reparative dentin forms by new odontoblasts. It is separated from the dentin by a calciotraumatic line
2. The rate of deposition depends on the degree of injury; the more severe the injury, the more rapid the rate of dentin deposition. As a result of this rapid deposition, cells often become trapped in the newly formed matrix, and the tubular pattern becomes grossly distorted

Question 21

Pulp Stones/Denticles

Answer 21

1. Mineralized mass within pulp
2. If they consist of dentin they are true stones or denticles
3. If they are NOT dentin, they are false stones
4. Stones may be attached to the dentin or free in the pulp cavity
5. Formation of pulp stones
   
   1. May be formed from trauma of operative procedures. Mineralization may occur around bits of dentin broken and scattered within the pulp
   2. May be formed from blood clots or around dead cells
   3. May be formed as diffuse calcifications of collagen fibers
6. Clinical significance of pulp stones
   
   1. They are not a source of infection
   2. They are of little clinical significance
   3. They may pose problems during endodontic procedures in that they make instrumentation of the pulpal chamber and root canals more difficult

Question 22

Etching dentin

Answer 22

1. Acid etching opens tubules by removing smear and sclerotic dentin
2. Hypochlorite solutions remove organic debris
3. Etching increases dentin permeability and acids are potentially damaging to the pulp

Question 23

Dentin permeability

Answer 23

1. The diameter of the tubules
   
   1. Larger tubules are more permeable; narrower sclerotic tubules are less permeable
2. The density of the tubules
   
   1. Increasing numbers of tubules increase permeability
3. Presence of occluding material
   
   1. Smear or sclerosis decreases dentin permeability
4. Thickness of dentin
   
   1. Distance the diffusing substance has to travel is a factor in permeability

Question 24

Three theories of tooth sensitivity

Answer 24

1. The dentin contains nerve endings that respond when it is stimulated
   
   1. some nerves occur within some tubules in the inner dentin but that dentin sensitivity does not depend solely, if at all, on the stimulation of such nerve endings
2. The odontoblasts serve as receptors and are coupled to nerves in the pulp
3. The tubular nature of dentin permits fluid movement to occur within the tubule when a stimulus is applied, a movement registered by pulpal free nerve endings close to the odontoblasts
4. The prevailing theory is that the sensitivity is caused by fluid flow through the dentinal tubues and transmitted via mechanoreceptors either inside the tubules or in the odontoblast layer
   
   1. The common treatment - using toothpastes containing inorganic compounds that desensitize the neurons and mineralize and seal the tubules.

Question 25

Dentin composition

Answer 25

1. Composition of bone and dentin are fairly similar
   
   1. ~70% mineral, ~20% organic, and rest water
2. Minerals:
   
   1. Crystalline - have a long range order, minimal repeating unit of a crystal called unit cell; mature mineral is crystalline apatitic calcium phosphate
   2. Amorphous - do NOT have a long range order
      
      1. Ionic - e.g. Amorphous Calcium Phosphate (ACP)
      2. Polymeric - e.g. Silica
   3. Minerals found in bone and denin are ionic crystallines
3. Hydroxyapatite unit cell is composed of 1) phosphate ions, 2) calcium ions, and 3) hydroxyl ions
4. Our body does NOT have stoichiometric hydroxyapatite; instead nonstochiometric carbonated hydroxyapatite is a major mineral phase in the mature mineralized tissues including dentin, bone, enamel, cementum and mineralized cartilage.
   
   1. A-type substitution - OH to CO3
   2. B-type substitution - PO4 to CO3
   3. Substitutions induce strain into the mineral lattice
5. Apatitic crystallites of dentin and bones are the smallest biogenic crystals known
   
   1. This has important implications since many mineral properties at nano-scale become qualitatively different from those at the macroscale
   2. Size, thickness and crystallinity of the bone mineral particles increase with age
   3. 2-6nm width, 30-50 nm length, 50-100 nm height

Question 26

Other mineral phases: transient metastable calcium phosphates

Answer 26

1. Amorphous Calcium phosphate is the most soluble and implicated for the prevention of caries
2. Hydroxyapatite (stoichiometric) is the least soluble
   
   1. Hydroxyapatite is the most thermodynamically stable calcium phosphate phase

Question 27

Collagen Type I

Answer 27

1. Major protein of bone and dentin ( and most abundant)
2. Mineralized collagen fibril is a basic building block of bone and dentin
3. Essential role in mineralization and mechanical properties
4. Type I Collagen is composed of triple helis
   
   1. Two alpha 1 chain
   2. One alpha 2 chain
5. ~1000 AA long peptide chains comprised of Gly-Pro-HPr repeating motif
   
   1. Glycine is always there
   2. Proline is the most frequent second AA
   3. Hydroxyprolin is the most frequent third AA
6. Glycine has great degree of freedom where as proline and hydroxyproline (with additional hydroxyl group) have limited rotational freedom
   
   1. Proline and hydroxyproline are what’s called iminine with bulky ring

Question 28

Amino acid composition of collagen

Answer 28

1. Glycine
   
   1. Smallest and simplest amino acid
   2. Extremely labile due to the absence of the side chain
2. Proline
   
   1. Only cyclic amino acid, formally imino acid due to the ring; due to the ring, has a rigid dihedral angle around -75 degree
3. Hydroxyproline
   
   1. Post-translational modification of proline, found mainly in collagen
   2. Stabilizes collagen molecules via hydrogen bonds and stereoelectronic effects
   3. Hydroxylation of proline by prolyl hydroxylase, requires ascorbic acid
4. Lack of ascorbic acid leads to collagen deficiency i.e. Scurvy
   
   1. Collagen production is compromised, leading to disintegration of connective tissues.
   2. Scorbutic gums is one of the symptoms; excessive bleeding and loss of teeth
   3. Consumption of garlic is easy fix

Question 29

Collagen triple helix

Answer 29

1. sequences with high concentration of prolines/hydroxyprolines tend to form polyproline helices
2. Collagen triple helix is comprised of 3 Polyproline type II helixes
3. Collagen molecules are ~300nm long and 1.5nm thick
4. Collagen Type I triple helix is stabilized by peptide-peptide and peptide-water hydrogen bonds
   
   1. I.e. hydrogen bonding between different chains and within same chain
5. Collagen fibrogenesis (refer to Lecture I)
6. Axial structure of collagen fibrils
   
   1. 0.4D overlap
   2. 0.6D gap
   3. Repeat of 67 nm
7. The staining and banding patterns are due to distribution of negatively charged regions that overlap
   
   1. The staining is stronger in polar, negatively charged regions

Question 30

Osteogenesis Imperfecta/Dentinogenesis Imperfecta Type I

Answer 30

1. The major cause of the osteogenesis imperfecta is destabilization of collagen triple helices, due to the substitution of obligatory Glycine in Gly-Xxx-Yyy motifs with other amino acids
   
   1. Depending on types of mutations, there are varying degree of severity
2. These conditions prevent proper assembly of collagen fibers into triple helix, thus shape of collagen fibers are compromised

Question 31

Acidic proteins of bone and dentin

Answer 31

1. Rich in Aspartic acid (Asp), Glutamic acid(Glu), and Serine (Ser), <–which can be phosphorylated
   
   1. 80% of serine is phosphorylated
2. Expressed in bones and dentin (the expression patterns as well as their relative quantities and distributions are different in different tissues)
3. COntrol nucelation, shape and organization of mineral crystals
4. Affect mechanical properties of bones and detin
5. DPP is the most negatively charged protein in human body
   
   1. 33% Asp, 58.8% Serine, 1.5% Glu

Question 32

Major Non-collagenous acidic proteins of dentin

Answer 32

1. SIBLING Family of proteins
   
   1. Osteopontin (OPN)
   2. Denin Matrix Protein1 (DMP1; AG1)
   3. Dentin Sialophosphoprotein (DSPP)
      
      1. Dentin Sialoprotein (DSP)
      2. Dentin Phosphoprotein (DPP, phosphophoryn)
      3. Dentin GLycoprotein (DGP)
2. Non-SIBLING proteins
   
   1. Osteocalcin (OC, bone gla protein)
      
      1. Gla= Gamma-carboxyglutamic acid
      2. Vitamin K dependent
      3. Gla modulates/inhibits mineralization
   2. Osteonectin (ON)
3. Non-collagenous proteins have multiple functions in biomineralization including formation of crystals, orientation of crystal growth, crystal proliferation, crystal maturation, etc.
   
   1. Pretty much all mineralization processes are modulatd by non-collagenous proteins
   2. The functions of the non-collagenous proteins in mineralization differ depending on the reaction conditions

Question 33

Small Integrin-Binding Ligand N-linked Glycoproteins (SIBLING)

Answer 33

1. Major proteins in bone and dentin
2. Acidic, rich is Asp, Glu, and Ser-P
3. Located on Chromosome 4, form a cluster at 4q13-4q21
4. Shar similar origin and similar properties
5. Common features:
   
   1. Both DSPP and DMP1 has its N-terminal portion highly glycosylated (but little phosphorylation [DSP]) whereas C-terminal portion highly phosphorylated (with little glycosylation [DPP])
   2. Both go through essential cleavages of the pro-proteins for proper functioning

Question 34

Mutations in DSPP

Answer 34

1. Mutations in DSPP cause dentinogenesis imperfecta (DI)
2. DSPP KO has a severe dental phoenotype similar to DI
   
   1. Type II and III DI also realted to mutations in DSPP
   2. Wider pulp, thinner dentin, lots of globular dentin, pulpitis, enamel worn off quickly, etc.
   3. Reduction in dentin thickness
   4. Thick predentin/hypomineralization; extensive globular dentin propagating throughout the dentin
   5. Very wide predentin layer

Question 35

Lack of DMP1

Answer 35

1. Similar phenotype to DSPP mutation
   
   1. Hypomineralization
   2. Widened pulp cavity
   3. Reduction in dentin thickness
   4. Lack of peritubular dentin
2. Role of DMP 1 in phosphate homeostasis
   
   1. DMP1 regulates expression of FGF23 which is involved in Pi metabolism
   2. In the absence of DMP 1, osteocytes typically capable of regulating FGF23 level is unable to do so, where FGF23 removes Pi in blood; phosphates are NOT kept in the body and not properly stored (due to too high FGF23 expression)
   3. This leads to rickets and thus can’t build proper bone due to phosphate deficiency

Question 36

Dentin Mineralization

Answer 36

1. Nonmineralized collagenous matrix is deposited by odontoblasts but mineralizes several microns away from the odontoblast layer i.e. predentin preceeds dentin
2. Collagen fibirls undergo structural changes prior to mineralization; proximal, central, to distal predentin
   
   1. Increase in space filling 30% to 70% to 100%
   2. Increase in fibril diameter ~30nm to 50-70nm to ~150nm
   3. Increase in D-spacing
3. Two pathways of transport of macromolecules and inorganic ions into predentin:
   
   1. Leaky junctions of odontoblasts directly seaping the materials through to predentin including PGs, GAGs, collagen, P, K, and Na
   2. Channels throughout odontoblast processes allowing for passage for phosphophoryn, Ca, PGs, GAGs; processing and removal of the predentin macromolecules
4. DPP (phosphophoryn) is secreted by odontoblast processes into predentin right at the mineralization front in its phosphorylated form, suggesting its possible role in mineralization
   5.

Question 37

Expression of major matrix proteins of dentin and enamel

Answer 37

1. Dentin proteins are transiently expressed by ameloblasts e.g. DSPP
2. Collagen, DSPP and Osteocalcin are all highly expressed in presecretory, secretory, and maturation odontoblasts
3. Osteonectin (ON) more sparsely distributed than osteocalcin (OC) in dentin

Question 38

Matrix vesicles

Answer 38

1. First mineral deposits in forming bone and dentin appear in matrix vesicles (MV)
2. MVs were also observed in bone repair sites
3. It is believed that MVs are essential for mineralization although exact mechanisms of their actions are unclear
4. Principle components of MVs include:
   
   1. Enzymes (including alkaline phosphatase)
   2. Transport proteins
   3. Integrins
   4. Lipids
5. Initial mineralization of mantle dentin starts in matrix vesicles (recall last topic)
   
   1. Matrix vesicles arise by budding from odontoblast processes
   2. Matrix vesicles contain amorphous calcium phosphate, which transforms into crystalline mineral and triggers mineralization of collagen fibrils.
   3. A similar process occurs in cememtum, cartilage, and bone
6. Mineral phase in matrix vesicles is amorphous calcium phosphate (ACP).
7. MVs are produced by osteoblasts, osteocytes, and odontoblasts
8. Possible mechanisms for the initiation of MVs induced mineralization
   
   1. MVs initially regulate ionic concentration across membranes of odontoblasts
   2. Nucleation (“crystallization”) of mineral crystals which migrate into the collagen fibrils
   3. MVs directly interact with collagen fibrils and initiate mineralization in a cooperative fashion

Question 39

Circumpalpal dentin

Answer 39

1. Mineralization of circumpapal dentin, including peritubular or intertubular dentin occurs at the mineralization front i.e. predentin/dentin boundary

Question 40

Mineralized collagen fibril

Answer 40

1. Mineralized collagen fibril is the basic building block of bone and dentin
2. Mineral crystals deposit inside collagen fibrils in the gaps and thus are separated by collagen molecules
3. Since more mineral is located in the gap regions, the mineralized fibrils have a bnaded appearance with periodicity matching D-spacing of collagen fibrils
4. Mutations in collagen cause defects of the fibrils, leading to abnormal mineralization, i.e. osteogenesis imperfecta (OI), suggesting that the structural organization of collagen fibrils is essential for proper mineralization
5. Large mineral crystals and acidic macromolecules (proteins) reside between collagen fibrils